We screened 10 Actinoplanes and 14 Dactylosporangium strains to investigate the biocatalytic ability of two genera of the Micromonosporaceae family. Two Actinoplanes strains (A. ferrugineus NBRC15555 and A. missouriensis NBRC102363) exhibited good growth when cultured in 228 and 231 media, as did two Dactylosporangium strains (Dactylosporangium sp. NBRC101297 and Dactylosporangium sp. NBRC101730) when cultured in 227 and 266 media. The stereoselective reduction of various carbonyl compounds using these four strains was therefore investigated. The present study discovered that these strains can reduce aliphatic and aromatic α-keto esters and an aromatic α-keto amide. On the basis of the conversion ratio and stereoselectivity of the alcohols produced, A. ferrugineus NBRC15555 is a potential biocatalyst for the stereoselective reduction of α-keto esters and an aromatic α-keto amide to the corresponding chiral alcohols when cultured in the 227 medium. Our results also suggest that the reduction of ethyl 2-methylacetoacetate by Dactylosporangium sp. NBRC101730 cultured in 227 medium in the presence of D-glucose is useful for the production of chiral ethyl 3-hydroxy-2-methylbutanoate.
Int.J.Curr.Microbiol.App.Sci (2019) 8(4): 920-931 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume Number 04 (2019) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2019.804.106 Biocatalytic Reduction of Carbonyl Compounds by Actinobacteria from Two Genera of the Micromonosporaceae Family: Actinoplanes and Dactylosporangium K Ishihara1*, K Morita1, Y Nishimori1, S Okamoto1, T Hiramatsu1, A Ohkawa1, D Uesugi2, M Yanagi1, H Hamada1, N Masuoka3 and N Nakajima4 Department of Life Science, Okayama University of Science, Okayama, Japan Department of Research & Development, JO Cosmetics Co., Ltd., Tokyo, Japan Department of Research & Development, Institute for Fruit Juice Research in Tsudaka, Co., Ltd., Okayama, Japan Department of Nutritional Science, Okayama Prefectural University, Soja, Okayama, Japan *Corresponding author ABSTRACT Keywords Biocatalyst, Actinomycete, Stereoselective reduction, Chiral hydroxy ester, Actinoplanes, Dactylosporangium Article Info Accepted: 10 March 2019 Available Online: 10 April 2019 We screened 10 Actinoplanes and 14 Dactylosporangium strains to investigate the biocatalytic ability of two genera of the Micromonosporaceae family Two Actinoplanes strains (A ferrugineus NBRC15555 and A missouriensis NBRC102363) exhibited good growth when cultured in 228 and 231 media, as did two Dactylosporangium strains (Dactylosporangium sp NBRC101297 and Dactylosporangium sp NBRC101730) when cultured in 227 and 266 media The stereoselective reduction of various carbonyl compounds using these four strains was therefore investigated The present study discovered that these strains can reduce aliphatic and aromatic α-keto esters and an aromatic α-keto amide On the basis of the conversion ratio and stereoselectivity of the alcohols produced, A ferrugineus NBRC15555 is a potential biocatalyst for the stereoselective reduction of α-keto esters and an aromatic α-keto amide to the corresponding chiral alcohols when cultured in the 227 medium Our results also suggest that the reduction of ethyl 2-methylacetoacetate by Dactylosporangium sp NBRC101730 cultured in 227 medium in the presence of D-glucose is useful for the production of chiral ethyl 3-hydroxy-2-methylbutanoate the class Actinobacteria, have been isolated from diverse habitats including soil, sediments, fresh and marine water, the rhizosphere, and plant tissues Several species belonging to Micromonosporaceae produce useful enzymes (Peczyňska-Czoch and Mordarski, 1988) and degradea variety of polysaccharides (Yeager et al., 2017) to Introduction Actinobacteria are among the most morphologically diverse prokaryotes, and are widely distributed in both terrestrial and aquatic ecosystems (Servin et al., 2007; Embley and Stackebrandt, 1994) Micromonosporaceae, a family of bacteria of 920 Int.J.Curr.Microbiol.App.Sci (2019) 8(4): 920-931 Industries Ltd (Japan) Bacto™ malt extract, Bacto™ yeast extract, and Difco™ soluble starch were purchased from Becton Dickinson and Co (USA) Ethyl lactate (2a), ethyl 3methyl-2-oxobutanoate (1f), ethyl 2-oxo-4phenylbutanoate (1h), ethyl 2-hydroxy-4phenylbutanoate (2h), and beef extract were purchased from Sigma-Aldrich Inc Ethyl benzoylformate (1g), ethyl 2methylacetoacetate (1j), and ethyl mandelate (2g) were obtained from Tokyo Chemical Industry, Co., Ltd (Japan) Ethyl 2oxobutanoate (1b), ethyl 2-oxopentanoate (1c), ethyl 2-oxohexanoate (1d), ethyl 2oxoheptanoate (1e), 2chlorobenzoylformamide (1i), 2chloromandelamide (2i), β-hydroxy esters (2b-f), and ethyl 3-hydroxy-2methylbutanoate (2j) were prepared as described previously (Mitsuhashi and Yamamoto, 2005; Kawai et al., 1995; Nakamura et al., 1988) All the other chemicals used in this study were of analytical grade and commercially available produce useful secondary metabolites (AlGarni et al., 2014; Solecka et al., 2012; Bérdy, 2005; Shomura et al., 1983) These bacteria therefore have important applications in industry, biotechnology, and agriculture (de Menezes et al., 2008; Rose and Steinbüchel, 2005; Linos et al., 2000) In addition, some strains of the genera Micromonospora and Salinispora in this family are useful biocatalysts for the asymmetric reduction of various carbonyl compounds such as α- and β-keto esters and aromatic α-keto amides (Ishihara et al., 2013, 2011) Thus, although these genera have thus been extensively studied for their biocatalytic activities, the potential ability of other genera in this family to serve as biocatalysts has not been investigated In this study, we investigated the stereoselective reduction of carbonyl compounds by members of two genera, Actinoplanes (Stackebrandt and Kroppenstedt, 1987; Couch, 1950) and Dactylosporangium (Thiemann et al., 1967), of the Micromonosporaceae family in order to identify potential novel biocatalysts (Figure 1) Microorganisms and Culture Actinoplanes italicus NBRC13911, Actinoplanes brasiliensis NBRC13938, Actinoplanes garbadinensis NBRC13995, Actinoplanes nipponensis NBRC14063, Actinoplanes violaceus NBRC14458, Actinoplanes ferrugineus NBRC15555, Actinoplanes capillaceus NBRC16408, Actinoplanes missouriensis NBRC102363, Actinoplanes rishiriensis NBRC108556, Actinoplanes siamensis NBRC109076, Materials and Methods Instruments and chemicals Gas chromatography (GC) was performed using a GL Science GC-353 gas chromatograph (GL Science Inc., Japan) equipped with capillary columns (DB-WAX, 0.25 µm, 0.25 mm x 30 m, Agilent Technologies, USA; TC-1,0.25µm, 0.25 mm x 30 m, GL Science Inc.; CP-Chirasil-DEX CB, 0.25 µm, 0.25 mm x 25 m, Varian Inc., USA; Gamma DEX 225, 0.25 µm, 0.25 mm x 30 m, Sigma-Aldrich Inc., USA) Ethyl pyruvate (Figure 1, 1a), diatomaceous earth (granular), and NZ amine, type A were purchased from Wako Pure Chemical Dactylosporangium salmoneum NBRC14103, Dactylosporangium vinaceum NBRC14181, Dactylosporangium matsuzakiense NBRC14259, Dactylosporangium rosum NBRC14352, Dactylosporangium fulvum NBRC14381, Dactylosporangium sp NBRC101297, Dactylosporangum sp NBRC101672, 921 Int.J.Curr.Microbiol.App.Sci (2019) 8(4): 920-931 Reduction of α- and β-keto esters, and an aromatic α-keto amide using resting actinomycete cells Dactylosporangium sp NBRC101673, Dactylosporangium sp NBRC101730, Dactylosporangium siamense NBRC106093, Dactylosporangium maewongense NBRC106094, Dactylosporangium darangshiense NBRC109065, Dactylosporangium tropicum NBRC109071, and Dactylosporangium luridum NBRC109093 Saline-washed wet Actinomycete cells (0.5 g, dry weight approximately 0.15 g) were resuspended in a arge test tube ( 30 mm x 200 mm) containing 20 mL of saline The substrate (0.15 mmol; 7.5 mM) was then added, and the reaction mixture was incubated aerobically (with reciprocated shaking at 120 rpm) at 25°C A portion (0.5 mL) of the mixture was applied to a short diatomaceous earth column ( 10 mm x 30 mm), extracted with diethyl ether (5.0 mL), and then concentrated under reduced pressure The above strains were purchased from the National Institute of Technology and Evaluation, Biological Resource Center (NBRC, Japan) These strains were maintained at 28°C in NBRC-recommended media (227, 228, 231, and 266) solidified with 1.5% (w/v) agar The 227 medium (International Streptomyces Project, ISP medium No 2) contained 4.0 g of Bacto™ yeast extract, 10.0 g of Bacto™ malt extract, and 4.0 g of D-glucose per liter of distilled water (pH 7.3) Analysis The production of alcohols (Figure 1, 2aj)was measured using a GC with a DB-WAX capillary column (100 kPa He at 110°C: 1a, 3.78 min; 2a, 4.75 min; 1b, 4.73 min; 2b, 5.92 min; 1f, 4.54 min; 2f, 6.41 min; 120°C:1c, 4.84 min; 2c, 6.45 min;1j, 5.54 min; 2j-anti, 7.62 min;2j-syn, 8.13 min; 150°C: 1d, 3.83 min; 2d, 4.68 min; 1e, 4.78 min; 2e, 6.07 min; 180°C: 1g, 9.01 min; 2g, 12.08 min) or a TC1 capillary column (100 kPa He at 140°C: 1h, 10.02 min; 2h, 10.96 min; 170°C: 1i, 6.85 min; 2i, 8.34 min) The enantiomeric excess (e.e.) of the product was measured using a GC instrument equipped with an optically active CP-Chirasil-DEX CB (2a-e, 2g-h, and 2j) or a Gamma DEX 225 capillary column (2f and 2i) The e.e was calculated using the following formula: e.e (%)= { (R-S)/ (R+S)} x 100, where R and S are the respective peak areas of the isomer in GC analyses The absolute configurations of the α- and βhydroxy esters (2a-h and 2j), and the αhydroxy amide (2i) were identified by comparing their retention times as determined by the GC analyses with those of authentic samples (Mitsuhashi and Yamamoto, 2005; Kawai et al., 1995; Nakamura et al., 1988) The 228 medium contained1.0 g of Bacto™ yeast extract, 1.0 g of beef extract, 2.0 g of NZ amine, type A, and 10.0 g of D-glucose per liter of distilled water (pH 7.3) The 231 medium contained 1.0 g of Bacto™ yeast extract, 1.0 g of beef extract, 2.0 g of NZ amine, type A, and 10.0 g of maltose per liter of distilled water (pH 7.3) The 266 medium contained 2.0 g of Bacto™ yeast extract, and 10.0 g of Difco™ soluble starch per liter of distilled water (pH 7.3) The 10Actinoplanes strains were grown in 227, 228, 231, and 266 media for days at 25°C with aerobic shaking in baffled flasks in the dark, and the 14 Dactylosporangium strains were grown in 227 and 266 media for days at 25°C with aerobic shaking in baffled flasks in the dark The actinomycetes were harvested by filtration on filter paper (Whatman No 4) in vacuo and washed with saline (0.85% NaCl aq.) The harvested cells were immediately used for reduction after washing with the saline 922 Int.J.Curr.Microbiol.App.Sci (2019) 8(4): 920-931 Results and Discussion Reduction of carbonyl compounds by Actinoplanes wet cells Screening of actinomycete strains and culture media Two actinomycete strains (A ferrugineus NBRC15555 and A missouriensis NBRC102363) that were cultivated in three media (227, 228, and 231) were tested for their ability to reduce several carbonyl compounds (Figure 1) The results of the microbial reductions are summarized in Table Both strains could reduce aliphatic and aromatic α-keto esters (1a-h) and an aromatic α-keto amide (1i) However, there were differences in the reduction rate and stereo selectivity of the alcohols produced that were dependent on the culture medium The reduction rate of substrates by NBRC15555 wet cells tended to be slightly higher when compared with NBRC1023063 wet cells More specifically, the reduction by NBRC15555 wet cells in 227 medium produced reduction ratios of 70% or more for all nine substrates tested We therefore tried to improve the conversion ratio and the stereo selectivity of the alcohols produced by the NBRC15555 strain by introducing additives into the reduction reaction catalyzed by NBRC15555 wet cells cultured in the 227 medium (see Table 4) Three additives were tested (glucose, sodium citrate, and soy oil), and considerable improvement was observed, especially with the addition of sodium citrate, with conversion ratios to >99% for all substrates Furthermore, four substrates (1a, 1c-e) were stereo specifically reduced to an e.e >99%, and the other substrates were converted to an e.e of nearly 90%.The reduction of 2-chlorobenzoylformamide (1i), an aromatic α-keto amide, demonstrated high stereo selectivity with all the wet cells tested (Figure 1) In particular, both the NBRC15555 and NBRC102363 wet cells cultured in228 medium reduced 1i to (R)-2i with a high conversion ratio and excellent stereo selectivity (>99% e.e.) As shown in Table 5, the reduction of ethyl 2- To determine the suitable media for liquid culture, 10 Actinoplanes and 14 Dactylosporangium strains were cultivated in several culture media, after which the wet weight of the cells was measured All Actinoplanes strains grew poorly in the 266 medium, even after days of culture, and the resulting wet cell weights were 0.1 g or less (see Table 1) However, two strains, Actinoplanes ferrugineus NBRC15555 and Actinoplanes missouriensis NBRC102363, yielded more than 0.5 g of wet cells/100 mL of culture in both 228 and 231 media, even though the recommended medium for NBRC15555 strain is 266 medium (Table 2) These results suggest that the amount of available carbon is more important than the type of carbon in liquid cultures of Actinoplanes strains Dactylosporangium strains exhibited good growth in liquid culture The amount of wet cells obtained when culturing in 227 medium, which contains glucose as a carbon source, was larger than when using 266 medium, which contains soluble starch Two strains in particular, Dactylosporangium sp NBRC101297 and NBRC101730, produced up to 1.0 g wet cells/100 mL when cultured in both 227 and 266 media We therefore investigated the potential ability of two Actinoplanes strains (NBRC15555 and NBRC102363) and two Dactylosporangium strains (NBRC101297 and NBRC101730) to act as biocatalysts for the asymmetric reduction of carbonyl compounds 923 Int.J.Curr.Microbiol.App.Sci (2019) 8(4): 920-931 methylacetoacetate (1j), one of the β-keto esters, by the wet cells of NBRC15555 cultured in 231 medium resulted in a conversion ratio >99%; however, the stereo selectivity (syn/antiratio and e.e.) was low and was not improved by the introduction of additives These results indicate that the NBRC15555 strain cultured in 227 medium is a useful biocatalyst for the asymmetric reduction of carbonyl compounds such as α-keto esters and aromatic α-keto amides Table.1 The cultivation of Actinoplanes strains in several culture medium Scientific name Actinoplanes italicus Actinoplanes brasiliensis Actinoplanes garbadiensis Actinoplanes nipponensis Actinoplanes violaceus Actinoplanes ferrugineus Actinoplanes capillaceus Actinoplanes missouriensis Actinoplanes rishiriensis Actinoplanes siamensis NBRC number 13991 13938 13995 14063 14458 15555 16408 102363 108556 109076 Recomm medium1 227 medium3 0.1 0.2 0.3 0.4 0.1 0.3 0.3 0.3 0.2 0.1 227 227 227 231 227 266 227 231 228 227 Wet cell weight (g)2 228 231 medium medium3 0.3 0.1 0.1 0.4 0.1 0.3 0.1 0.2 99 >99 R 29 >99 R Substrate (0.15 mmol), 0.85% NaCl aq (20 mL) were added to the wet cells (0.5 g) cultured in liquid medium, and the reaction mixture was incubated aerobically (reciprocating shaking at120 min-1) at 25 °C for 48 hrs Conversion was measured by a GLC analysis Enantiomeric excess (e.e.) and absolute configuration (R/S) were determined by GLC analyses with optically active capillary columns 925 Int.J.Curr.Microbiol.App.Sci (2019) 8(4): 920-931 Table.4 The reduction of by A ferrugineus NBRC15555 cultivated in 227 medium in the presence of additive.1,2,3 D-Glucose Product Con (%) e.e (%) R/S 2a 2b 2c 2d 2e 2f 2g 2h 2i >99 59 62 74 30 >99 42 45 99 42 34 19 13 65 19 >99 15 >99 S R R R R S R R R Additive Sodium citrate Conv e.e (%) (%) R/S >99 >99 >99 >99 >99 >99 >99 >99 >99 >99 92 >99 >99 >99 99 90 88 >99 S R R S S S S S R Con v, (%) >99 >99 >99 95 90 >99 49 90 98 Soy oil e.e (%) R/S 43 60 97 40 18 35 43 43 >99 S S S S S S S S R Substrate (0.15 mmol), 0.85% NaCl aq (20 mL) and additive ( D-glucose and sodium citrate: mmol, soy oil: 0.5 mL) were added to the wet cells (0.5 g) cultured in 227 medium, and the reaction mixture was incubated aerobically (reciprocating shaking at120 -1) at 25 °C for 48 hrs Conversion was measured by a GLC analysis Enantiomeric excess (e.e.) and absolute configuration (R/S) were determined by GLC analyses with optically active capillary columns Table.5 The reduction of ethyl 2-methylacetoacetate (1j) to the corresponding alcohol (2j) by Actinoplanes strains.1,2,3 Strain A ferrugineus A missoriensis A ferrugineus medium Additive 227 228 231 227 228 231 227 227 227 None None None None None None D-Glucose Sodium citrate Soy oil Conv (%) 16 >99 95 10 12 Syn / Anti 20 / 80 44 / 56 59 / 41 38 / 62 41 / 59 35 / 65 33 / 67 22 / 78 39 / 61 e.e (%) Syn-(2R, Anti-(2S, 3S) 3S) 33 92 38 45 34 17 30 78 20 31 42 84 40 80 31 88 44 71 Substrate (0.15 mmol), 0.85% NaCl aq (20 mL) and additive ( D-glucose and sodium citrate: mmol, soy oil: 0.5 mL) were added to the wet cells (0.5 g) cultured in liquid medium, and the reaction mixture was incubated aerobically (reciprocating shaking at120 min-1) at 25 °C for 48 hrs Conversion was measured by a GLC analysis Enantiomeric excess (e.e.) and absolute configuration (R/S) were determined by GLC analyses with optically active capillary columns 926 Int.J.Curr.Microbiol.App.Sci (2019) 8(4): 920-931 Table.6 The reduction of ethyl 2-methylacetate (1j) to the corresponding alcohol (2j) by two Dactylosporangium strains.1,2,3 Product 2a 2b 2c 2d 2e 2f 2g 2h 2i Dactylosporangium sp NBRC101297 227 medium 266 medium Conv e.e Conv e.e (%) (%) R/S (%) R/S (%) 40 87 S 51 79 S 29 53 S 95 76 S 23 42 S 74 37 S 14 23 S >99 S 14 S S 32 66 S 60 14 S 11 12 R 10 25 R 80 S >99 S 25 25 R 94 85 R Dactylosporangium sp NBRC101730 227 medium 266 medium Conv e.e Conv e.e (%) R/S (%) R/S (%) (%) 94 47 S 90 22 S 48 18 S 43 24 S 46 14 S 35 33 S 36 40 S 29 19 S 14 36 S >99 S 69 >99 R 75 22 R 26 64 S 19 47 S 16 39 S 28 S 80 38 R 31 53 R Substrate (0.15 mmol), 0.85% NaCl aq (20 mL) were added to the wet cells (0.5 g) cultured in liquid medium, and the reaction mixture was incubated aerobically (reciprocating shaking at120 -1) at 25 °C for 48 hrs Conversion was measured by a GLC analysis Enantiomeric excess (e.e.) and absolute configuration (R/S) were determined by GLC analyses with optically active capillary columns Table.7 The reduction of carbonyl compounds (1a-i) to the corresponding alcohols (2a-i) by Dactylosporangium sp NBRC101297 strain in the presence of additive.1,2,3 227 medium D-Glucose Product 2a 2b 2c 2d 2e 2f 2g 2h 2i Conv (%) 48 53 42 23 14 66 12 48 e.e (%) 83 73 81 >99 >99 46 48 83 266 medium L-Glutamate R/S S S S S S S R S R Conv (%) 76 68 68 32 60 80 22 35 38 e.e (%) 43 78 81 72 >99 28 46 58 60 D-Glucose R/S S S S S S R R S R Conv (%) 15 29 33 11 25 52 18 14 49 e.e (%) 72 64 74 63 >99 21 45 46 88 L-Glutamate R/S S S S S S S S S R Conv (%) >99 82 92 64 90 34 34 41 e.e (%) >99 69 68 68 54 30 54 45 80 Substrate (0.15 mmol), 0.85% NaCl aq (20 mL) and additive (glucose and sodium hydrogen glutamate: mmol) were added to the wet cells (0.5 g) cultured in liquid medium, and the reaction mixture was incubated aerobically (reciprocating shaking at120 min-1) at 25 °C for 48 hrs Conversion was measured by a GLC analysis Enantiomeric excess (e.e.) and absolute configuration (R/S) were determined by GLC analyses with optically active capillary columns 927 R/S S S S S S R R S R Int.J.Curr.Microbiol.App.Sci (2019) 8(4): 920-931 Table.8 The reduction of carbonyl compounds (1a-i) to the corresponding alcohols (2a-i) by Dactylosporangium sp NBRC101730 strain in the presence of additive.1,2,3 227 medium D-Glucose Product 2a 2b 2c 2d 2e 2f 2g 2h 2i Conv (%) 71 25 57 45 18 71 11 >99 e.e (%) 55 >99 40 55 13 20 67 10 91 R/S S S S S S S R R R 266 medium L-Glutamate Conv e.e (%) (%) R/S >99 49 S 62 58 S 32 36 S 39 39 S 46 S >99 S 77 S 44 S 95 27 R D-Glucose Conv (%) 71 59 64 58 25 >99 28 31 47 e.e (%) 36 45 27 22 >99 18 67 42 90 R/S S S S S S R R S R L-Glutamate Conv e.e (%) (%) R/S 79 54 S 25 55 S 57 22 S 39 33 S 36 >99 S 71 75 R 32 81 R 13 48 S 41 80 R Substrate (0.15 mmol), 0.85% NaCl aq (20 mL) and additive (glucose and sodium hydrogen glutamate: mmol) were added to the wet cells (0.5 g) cultured in liquid medium, and the reaction mixture was incubated aerobically (reciprocating shaking at120 min-1) at 25 °C for 48 hrs Conversion was measured by a GLC analysis Enantiomeric excess (e.e.) and absolute configuration (R/S) were determined by GLC analyses with optically active capillary columns Table.9 The reduction of ethyl 2-methylacetoacetate (1j) to the corresponding alcohol (2j) by two Dactylosporangium strains in the presence of additive.1,2,3 Strain Medium 227 Dactylosporangium sp NBRC101297 266 227 Dactylosporangium sp NBRC101730 266 Additive Conv (%) Syn / Anti None Glucose Glutamate None Glucose Glutamate None Glucose Glutamate None Glucose Glutamate 18 29 86 20 68 38 98 46 26 63 50 10/90 10/90 11 / 89 8/92 11/89 10 / 90 / 93 99 99 / 94 99 99 e.e (%) Syn-(2R, Anti-(2S, 3S) 3S) >99 >99 >99 >99 >99 >99 >99 >99 >99 >99 >99 >99 >99 >99 >99 -4 >99 -4 >99 >99 >99 -4 >99 -4 Substrate (0.15 mmol), 0.85% NaCl aq (20 mL) and additive (D-glucose and sodium hydrogen glutamate: mmol, soy oil: 0.5 mL) were added to the wet cells (0.5 g) cultured in liquid medium, and the reaction mixture was incubated aerobically (reciprocating shaking at120 min-1) at 25 °C for 48 hrs Conversion was measured by a GLC analysis Enantiomeric excess (e.e.) and absolute configuration (R/S) were determined by GLC analyses with optically active capillary columns -: E.e could not be measured because it could not detect the Anti-form 928 Int.J.Curr.Microbiol.App.Sci (2019) 8(4): 920-931 Fig.1 The reduction of various carbonyl compounds (1a-j) to the corresponding alcohols (2a-j) by actinomycetes Fig.2 The reduction of 1j to (2S, 3S)-2j by Dactylosporangium sp NBRC101730 strain in the presence of glucose OH OH CO2Et O (2R, 3S) Dactylosporangium strain CO2Et CO2Et Syn-2j OH 1j (2S, 3R) OH CO2Et (2R, 3R) CO2Et Anti-2j (2S, 3S) Conv.: 98% ratio and stereoselectivity of the alcohols produced The effects of additives on these microbial reduction reactions were also examined (Tables and 8) The NBRC101297 strain cultured in 266 medium in the presence of sodium hydrogen Lglutamate reduced ethyl pyruvate (1a) to the corresponding (S)-alcohol (2a) with a high conversion ratio (>99%) and excellent e.e (> 99%) However, the conversion rate and the Reduction of carbonyl compounds by Dactylosporangium wet cells Two Dactylosporangium strains cultivated in two media were tested for their ability to reduce several carbonyl compounds (Table 6) The Dactylosporangium strains could reduce aliphatic and aromatic α-keto esters and an aromatic α-keto amide, and no substantial difference was observed in the conversion 929 Int.J.Curr.Microbiol.App.Sci (2019) 8(4): 920-931 stereoselectivity of the reduction of other substrates by same strain were not greatly improved with additive (Table 7) Similarly, the reduction efficiency and stereoselectivity by the NBRC101730 strain were not improved with additives (Table 8) antimicrobial actinomycetes strains from Saudi environment J Food, Agric Environ 12: 1073-1079 Bérdy, J 2005 Bioactive microbial metabolites J Antibiot 58: 1-26 Couch, J.N 1950 Actinoplanes, a new genus of the Actinomycetales J Elisha Mitchell Sci Soc., 66: 87-92 de Menezes, A.B., Lockhart, R.J., Cox, M.J., Allison, H.E and McCarthy, A.J 2008 Cellulose degradation by micromonosporas recovered from freshwater lakes and classification of these actinomycetes by DNA gryase B gene sequencing Appl Environ Microbiol 74: 7080-7084 Embley, T.M and Stackerbrandt, E 1994 The molecular phylogeny and systematics of the actinomycetes Annu Rev Microbiol 48: 257-289 Ishihara, K., Nagai, H., Takahashi, K., Nishiyama, M and Nakajima, N 2011 Stereoselective reduction of βketo ester and β-keto amide with marine actinomycetes, Salinispora strains, as novel biocatalysts Biochem Insights 4: 29-33 Ishihara, K., Fujita, A., Sakiyama, A., Kobayashi, U., Hori, K., Maruike, K., Masuoka, N., Nakajima, N and Hamada, H 2013 Preparation of chiral hydroxy esters using actinobacteria: biocatalyst activity of marine-derived Micromonospora and Streptomyces strains Open J Appl Sci 3: 116-122 Kawai, Y., Takanobe, K and Ohno, A 1995 Stereochemical control in microbial reduction XXV Additives controlling diastereoselectivity in a microbial reduction of ethyl 2-methyl-3oxobutanoate Bull Chem Soc Jpn 68: 285-288 Linos, A., Berekaa, M.M., Reichelt, R., Keller, U., Scmitt, J Flemming, H.C., Kroppenstedt, R.M., Steinbüchel, A In contrast, the effect of additives on the reduction of β-keto ester was remarkable, and ethyl 2-methylacetoacetate (1j) reduction by NBRC101730 cultured in 227 medium in the presence of glucose stereospecifically produced the corresponding alcohol (2S, 3S)2j (Table 9) More specifically, in this reaction, the substrate was reduced to only one of the four theoretically possible isomers, in other words, this microbial reduction reaction was able to obtain a β-hydroxy ester having two chiral center carbons (Figure 2) In conclusion, members of two genera (Actinoplanes and Dactylosporangium) from the Micromonosporaceae family were shown toconvert various α-keto esters and an aromatic α-keto amide to the corresponding hydroxy esters and hydroxy amide On the basis of the conversion ratios and the stereoselectivity of the products, we suggest Actinoplanes ferrugineus NBRC15555 cultured in the 227 medium for potential use as a biocatalyst for the stereoselective reduction of α-keto esters and the α-keto amides to yield the corresponding chiral alcohols Our results also suggest that the reduction of ethyl 2-methylacetoacetate by Dactylosporangium sp NBRC101730 cultured in the 227 medium in the presence of D-glucose stereospecifically produces the corresponding chiral β-hydroxy ester References Al-Garni, S.M., Sabir, J.S.M., El Hanafy, A.A.E.M., Kabli, S.A., Al-Twiley, D.A and Ahmed, M.M 2014 Isolation and identification of 930 Int.J.Curr.Microbiol.App.Sci (2019) 8(4): 920-931 2000 Biodegradation of cis-1,4polyisoprene rubbers by distinct actinomycetes: microbial strategies and detailed surface analysis Appl Environ Microbiol 66: 1639-1645 Mann, J 2001 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Rajnisz, A 2012 Biologically active secondary metabolites from Actinomycetes Central Eur J Biol 7: 373-390 Stackebandt, E and Kroppenstedt, R.M 1987 Union of the genera Actinoplanes couch, Ampullariella couch, and Amorphosporangium couch in a redefined genus Actinoplanes Syst Appl Microbiol 9: 110-114 Thiemann, J.E., Pagani, H and Beretta, G (1967) A new genus of the Actinoplanaceae: Dactylosporangium, gen nov Archiv für Microbiologie 58: 42-52 How to cite this article: Ishihara, K., K Morita, Y Nishimori, S Okamoto, T Hiramatsu, A Ohkawa, D Uesugi, M Yanagi, H Hamada, N Masuoka and Nakajima, N 2019 Biocatalytic Reduction of Carbonyl Compounds by Actinobacteria from Two Genera of the Micromonosporaceae Family: Actinoplanes and Dactylosporangium Int.J.Curr.Microbiol.App.Sci 8(04): 920-931 doi: https://doi.org/10.20546/ijcmas.2019.804.106 931 ... H Hamada, N Masuoka and Nakajima, N 2019 Biocatalytic Reduction of Carbonyl Compounds by Actinobacteria from Two Genera of the Micromonosporaceae Family: Actinoplanes and Dactylosporangium Int.J.Curr.Microbiol.App.Sci... investigated the stereoselective reduction of carbonyl compounds by members of two genera, Actinoplanes (Stackebrandt and Kroppenstedt, 1987; Couch, 1950) and Dactylosporangium (Thiemann et al., 1967), of. .. rate and the Reduction of carbonyl compounds by Dactylosporangium wet cells Two Dactylosporangium strains cultivated in two media were tested for their ability to reduce several carbonyl compounds